Controlled Fab-arm exchange for the generation of stable bispecific IgG1

Nat Protoc. 2014 Oct;9(10):2450-63. doi: 10.1038/nprot.2014.169. Epub 2014 Sep 25.

Abstract

The generation of bispecific antibodies (bsAbs) with natural IgG architecture in a practical and efficient manner has been a longstanding challenge. Here we describe controlled Fab-arm exchange (cFAE), which is an easy-to-use method to generate bispecific IgG1 (bsIgG1). The protocol involves the following: (i) separate expression of two parental IgG1s containing single matching point mutations in the CH3 domain; (ii) mixing of parental IgG1s under permissive redox conditions in vitro to enable recombination of half-molecules; (iii) removal of the reductant to allow reoxidation of interchain disulfide bonds; and (iv) analysis of exchange efficiency and final product using chromatography-based or mass spectrometry (MS)-based methods. The protocol generates bsAbs with regular IgG architecture, characteristics and quality attributes both at bench scale (micrograms to milligrams) and at a mini-bioreactor scale (milligrams to grams) that is designed to model large-scale manufacturing (kilograms). Starting from good-quality purified proteins, exchange efficiencies of ≥95% can routinely be obtained within 2-3 d (including quality control).

MeSH terms

  • Antibodies, Bispecific / chemistry
  • Antibodies, Bispecific / genetics
  • Antibodies, Bispecific / metabolism*
  • Bioreactors
  • Genetic Vectors / genetics
  • Genetic Vectors / metabolism
  • HEK293 Cells
  • Humans
  • Immunoglobulin Fab Fragments / genetics
  • Immunoglobulin Fab Fragments / metabolism
  • Immunoglobulin G / genetics
  • Immunoglobulin G / metabolism*
  • Mass Spectrometry / methods
  • Oxidation-Reduction
  • Point Mutation
  • Protein Engineering / instrumentation
  • Protein Engineering / methods*
  • Quality Control

Substances

  • Antibodies, Bispecific
  • Immunoglobulin Fab Fragments
  • Immunoglobulin G